Project Summary Chlamydia trachomatis is the leading cause of preventable blindness and the most commonly acquired sexually transmitted infection of bacterial origin, with over 4 million new cases in the United States and an estimated 92 million cases worldwide each year. Importantly, there are substantial ethnic and gender disparities associated with chlamydial infection, where the majority of undiagnosed chronic infections are subclinical in women and linked to an increased risk for developing pelvic inflammatory disease, infertility, cervical cancer, stillbirths, and premature deliveries.While the precise mechanisms responsible for these clinical manifestations remain largely undetermined, we do know that Chlamydial infections require establishment and maintenance of a unique intracellular replication niche for completion of a complex biphasic lifecycle. However, many of the host factors involved in this process are not known and this gap in our knowledge limits our understanding of the C. trachomatis lifecycle and pathogenesis associated Chlamydia infections. The long-term objective of our research is to determine the molecular mechanisms that Chlamydia employs to establish an infection. We hypothesize that C. trachomatis relies on a subset of genes in the human genome for successful completion of the bacterial lifecycle and knock-outs of these host genes will perturb the Chlamydia lifecycle and prevent cell death. To test this hypothesis, we are using a CRISPR-Cas9 based high- throughput genome wide knock-out screen to identify novel genes and microRNAs that are resistant to C. trachomatis infection. Additionally, we will generate individual gene knock-outs to investigate the role of specific host genes in the cytoskeleton and vesicle transport pathways. Specifically, this proposal aims to (1) identify mutations in genes or microRNAs that are essential for the C. trachomatis lifecycle (2) knockout individual genes in specific host pathways and determine their role in C. trachomatis entry and inclusion germination and maturation.